CA1328239C - Bacillus thuringiensis strain, method for their isolation and related insecticidal compositions - Google Patents

Abstract

ABSTRACT An insecticidal composition comprising at least one of the bacteria selected from Bacillus thuringiensis strains identified as NRRL deposits under accession numbers as: The invention also provides a method for producing a Bacillus thuringiensis having selective insecticidal activity comprising: (a) providing a first Bacillus thuringiensis strain, having a specific insecticidal activity conferred by a gene coding for an insecticidal toxin protein, the gene being located on a plasmid, in admixture with an intermediate Bacillus re-cipient strain whereby the intermediate Bacillus recipient strain acquires by conjugation the plasmid conferring insec-ticidal activity; (b) isolating and identifying the intermediate Bacillus recipient strain which has acquired the plasmid conferring insecticidal activity; (c) providing the transconjugant intermediate Bacillus recipient strain isolated in step (b) in admixture with a second Bacillus thuringiensis strain whereby the second Bacillus thuringiensis strain acquires the plasmid conferring insecticidal activity from the transconjugant intermediate Bacillus recipient strain and (d) isolating and identifying a transconjugant from the culture admixture of step (c), having selectively targeted insecticidal activity.

Description

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1. INTRODUCTION
This invention relates to new strains of Bacillus `~ thuringiensis and a method for their isolation, identifica~ion and improvement. These new strains have enhanced activity against lepidopteran pests. This invention also relates to insecticidal compositions incorporating these novel strains.
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2. BACKGROUND OF THE INVENTION
2.1. CO~MERCIAL PESTICIDES: GENERA~ C_NSIDERATIONS
Each year, significant portions of the world's commercially imp~rtant agricultural crops are lost to insects and other pest infestation. The damage wrought by thesa pests extends to all areas of commercially important plants including foods, textiles, and various domestic plants, and the economic damage runs well into the millions of dollars. Thus, protection of crops from such ~i infastations is of paramount concern-Broad spectrum pesticides are most commonly used ~, for crop protection, but indiscriminate use of these agents ~i can lead to disruption of the plant's natural defensive j agents. Furthermore, because of their broad spectrum of activity, the chemical pesticides may destroy non-target organisms such as beneficial insects and parasites of destructive pests. These are also frequently toxic to ~l animals and humans, and thus, pose environmental hazards when applied.
Additionally, insects and other organisms have frequently developed resistance to these pesticides when rapeatedly exposed to them. In addition to raducing the utility of the pesticide, resistant strains of minor pests q may become major infestation problems due to the reduction - of beneficial parasitic organisms.
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` _4~ 8239 ` This is a major problem encountered in using broad spectrum pesticides. What is needed is a biodegradable pesticide that combines a narrower spectrum of activity with an ability o~ maintaining its activity over an extended period of time, i.e., to which resistance develops much more slowly, or not at all. Biopesticides appear to be useful in this regard.
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2.2. BIOLOGICA~ PESTICIDES
Biopesticides, also called biorationals, make use of naturally occurring pathogens (diseases) to control insect, ~ungal, and weed infestations of agricultural crops.
Such substances comprise a bacterium which produces a substance toxic to the infesting agent (a toxin), with or without a bacterial growth medium. Such bacteria can be applied directly to the plants by standard methods of application, and are typically less harmful to non-target organisms and the environment as a whole, in comparison to chemical pesticides.
The use of biological methods of pest control was first suggested in 1895 when a fungal disease was discovered in silkworms. It was not until 1940, however, when spores - of the milky disease bacterium Bacillus popilliae J applications were used to control the Japanese beetle, tha~
j 25 successful biological pest control was first achieved. A
bacterium named Bacillus thuringiensis (BT) that makes a toxin ~atal to caterpillars is currently the most widely used biopesticide. In the late 1960's, the discovery of HD-1, a highly toxic strain of BT, set the stage for commercial use of biopesticides.
~i ~; 2.3. Bacillus_thuringiensis ~ND_DELTA-ENDOTOXINS
Bacillus thuringiensis (otherwise known as ~B.t."
~;, or nBTN) is a widely distributed, rod shaped, aerobic and spore ~orming microorganism. During i~s sporulation cycle ,.

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:: , ~ 5~ ~328239 BT forms proteins known as protoxins or delta-endotoxins.
These protoxins are deposited in BT as parasporal, crystalline inclusions or as part of the spore coat. The pathoqenicity o~ BT to a variety of sensitive insects, such as those in the orders Lepidoptera and Diptera, is ~`` essentially due to this parasporal crystal, which may ; represent over 20~ of the dry weight of the BT cell at the time of sporulation.
The parasporal crystal is active in the insect only after ingestion. For instance, a~ter ingestion by a lepidopteran insect, the alkaline pH and proteolytic enzymes ~;~ in the mid-gut activate the crystal allowing the release of the toxic components. These toxic components poison the mid-gut cells causing the insect to cease feeding and eventually to die. In fact, BT has proven to be an effective and environmentally safe insecticide in dealing with lèpidopteran pests.
It has been reported that different strains of BT
produce serologically different parasporal crystals.
s 20 However, one of the predominant crystal forms, of bipyramidal shape, produced by many of the BT strains is composed of a protein(s) known as P1. Pl proteins have a molecular weight of about 130,000 (d) and may also be present in the spore coat. The genes for the parasporal ~` 25 crystal Pl, and those of most of the ot~er protein crystals, ; reside on one or more of a large number of plasmids of varying size in BT.

3. SUMM~RY OF INVENTION
This invention provides for biologically pure ; strains of acillus ~ which have insecticidal activity against insects of the order Lepidoptera. These strains have been derived by both plasmid curing and ` conjugation procedures.
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: It is also an object of this invention to provide . a novel method for recognizing the plasmid containing the gene coding for a toxin protein in a BT strain and, thereby, enable the selective use of ~pecific strains of BT for ~,Z 5 plasmid curing and conjugation experiments so as to derive a : strain of BT having sp~cific or enhanced insecticidal activity.
It is further an object o~ this invantion to provide a method for controlling insects in the order Lepidoptera with these novel Bacillus thuringiensis strains.
.. ~ All of the above e~bodimentZs o~ this invention will be-;` described in greater detail in the description of the ~ invention which follows.
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4. BRIEF DESCRIPTION OF THE FIGURES
FIGU~E 1 is a photograph of a gel electrophoresis of solubilized crystals from HD1-1 and several derived ~l strains, which shows dif~erential production of Pl and P2 Z crystal proteins in the various strains.
FIGURE 2 is a photograph o~ a gel electrophoresis ~ which shows the plasmid arrays of the novel BT strains ~ depoZsited with the NRRL and also BT strains used as donors ~, and recipients, as described in 5.1-5.8, and ~onstructed as ;Z illustrated in Figure 3.
~/ 25 FIGURE 3 is a flow chart illustrating the -~ construction o~ novel BT strains described in 5.1-5.8.
~ FIGURE 4 is a photograph of a gel electrophoresis ~ which shows the plasmid arrays of novel BT deposited with the NRRL, as well as BT strains used as donors and recipients, as described in 5.9-5.11, and constructed as illustrated in Figure 5.
'` FIGURE 5 is a flow chart illustrating the . construction of novel BT strains described in 5.~-5.11.

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5. DESCRIPTION OF THE INVENTION
: ~enerally stated, the present invention provides :: novel Bacillus thuringiensis strains which have insecticidal activity against insectæ of the order Lepidoptera.
Biologically pure cultures of these strains have been deposited with the NRRL. Bioassays described below have confirmed the activity of these strains. These strains of BT, therefore, are preferred for use as at least one of the active ingredients in an insecticidal composition useful against lepidopteran, dipteran, or coleopteran insects.
Essentially this invention comprises combining and optimizing several technigues (eOg., isolation of new BT
strains, curing and transfer o~ toxin plasmids, use of isogenic strains, plasmid array analysis, assigning specific i 15 toxicities to individual toxin plasmids), so as to achieve a novel systematic approach o~ modifying B.t. strains for greater toxicity to any given susceptible insect.
- Generally stated, this invention provides a . method for producing a Bacillus thuri~giensis having `~ 20 selective insecticidal activity against insects comprising:
(a) providing a first Bacillus thuringiensis ' strain, having a specific insecticidal activity conferred by :'. a gene coding for an insecticidal toxin protein, said gene ^, being located on a plasmid, in admixture with an intermediate Bacillus recipient strain whereby said r intermediate Bacillus recipient strain acguires by . conjugation the plasmid conferring insecticidal activity;
/ (b) isolating and identifying said intermediate : Bacillus recipient strain which has acquired said plasmid conferring insecticidal activity;
, (c) providing the transconjugant intermediate '!i~ Bacillus recipient strain isolated in step (b) in admixture , with a ~econd Bacillus thuringiensis strain whereby said s~
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: second Bacillus thuringiensis strain acquires the plasmid conferring insecticidal activity from said transconjugant intermediate Bacillus recipi~nt strain and ~d) isolating and identifying a transconjugant . 5 from the culture admixture of step (c), having selectively ~ targeted insecticidal activity.
- The method above also encompasses the embodiment ~. wherein the transconjugant of step (d) produces increased :~ amounts of insecticidal toxin over the said second Bacillus ~, 10 thuringiensis strain.
The method above additionally encompasses the ~ embodiment wherein identification of the transconjugants in steps (b) and (d) is by visualization of the plasmid arrays o~ the intermediate Bacillus recipient strain and the second Bacillus thuringiensis~
., For instance, in a preferred embodiment of this invention a first Bacillus thurin~iensis strain having, for example, lepidopteran activity, is provided in admixture ;~, first with a second Bacillus thuringiensis strain (or B.
,. 20 cereus) whereby said second Bacillus thuringiensis strain .~ acquires (by conjugation~ the plasmid conferring :. insecticidal activity against Lepidoptera. ~he strain which 'i acquired the toxin encoding plasmid is identified by m2thods ~ such as gel electrophoresis to determine its plasmid array : 25 which would show plasmids acquired, besides those known to exist in that second strain); isolating the strain which acquired the toxin plasmid and then providing that.
~Z~ transconjugant strain in admixture with a third Bacillus thurin~ is having a selective insecticidal activity (i.e.
.~, 30 to different lepidopteran insects or to diptera or ., coleoptera~ under conditions favoring aonjugation whereby ~i, said second Bacillus thuringiensis strain having activity :i ~' acquires the plasmid conferring insecticidal aativity by .~ conjugation from said transconjugant s~rain. The resultant X
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activity against different species of lepidopteran pests (each 9f which have varying degrees of sensitivity to a particular BT toxin), or again~t lepidopteran and dipteran insects, lepidopteran and coleopteran, or dipteran and coleopteran pests. The new BT strains of this invention may .:
serve as an inexhaustible source of toxin plasmids of greater specificity and toxicity, which can then be transferred by conjugation into any of several recipient strains to generate novel strains with previously unknown combinations of toxin plasmids and toxin proteins.
This invention also provides for novel insecticides for use against Lepidoptera, Coleoptera, or - Diptera comprising a mixture of BT and a suitable carriar.
The BT strain or strains may be used in the form of spores, whole organisms, or a combination of these. A suitable carrier may be any one of a number of solids or liquids known to those of skill in the art.
., All of these aspects of the invention are described below in detail and are illustrated in the following examples.
;, ,` 5.1. CURING OF B. THURINGIENSIS AND CONJUGATION
Insecticidal strains of Bacillus thuringiensis ~BT) are distinguished from the related species B~ cereus by their production of a proteinaceous inclusion, the 1 parasporal crystal, during sporulation. The protein(s) that '~ make up the crystal(s) determine the toxicity of the individual BT strain (that is, whether lepidopteran, dipteran, or coleopteran larvae are affected). The genes encoding the proteins of the toxin crystals are located on extrachromosomal DNA molacules (plasmids). BT strains making large amounts of toxin crystal protein have been ¦ shown by various technical approaches to contzin two or more ! distinct toxin plasmids. Each toxin plasmid in a strain codes for its own toxin protein(s), which can often be ., I , i!

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-` ~3~239 - distinguished from the toxin protein(s) encoded by the other toxin plasmid(s) present, by immunological, elec~rophoretic, or other technical means.
. Curing is the loss of plasmid DNA. Curing of oneor m~re toxin plasmids tin a multiple toxin plasmid BT
strain), and possibly even non-toxic plasmids, may lead to increased production of toxin protein(s) encoded by the remaining toxin plasmid(s~. If the remaining toxin plasmid or plasmid~ encode a mor~ potent toxin than did the lost toxin plasmid or plasmids, the toxicity of the derived, ~- partially-cured s~rain will be greater on a protein basis, :~ and sometimes also on a raw (dosage) basis. Thus, by curing a BT strain of specific plasmids, the type of toxin protein ~, that it synthesizes may be altered to give greater toxicity :~ 15 against a given target insect. This ~an mean that the toxin derivative would be more specific against that insect.
Curing of plasmids may be achieved by number of i different methods. Plasmid curing does occur spontaneously at a low level, and these spontaneously cured strains may b~
detected by routine screening. However, curing can also be actively induced, by elevation of the culture temperature.
~l This is preferably done in step~, i.e., progressivsly ,~ brought up from about 37C up to about 45C. Exposure of .~, the strains to detergents, such as sodiumdodecyl sulfate or ; 25 chemicals which interfere with DNA replication, such as .i acridines, ethidium bromide, or novobiocin, may also be used .~ to increase the frequency of plasmid curing. For the .1 present purposes 9 elevated temperature is generally I preferred. B~ toxin plasmids of a medium size range (about l 30 40 to 90 megadaltons ~d)) can usually transfer from the ;.~ strain that carries them into other BT or B. cereus strains.
~ I~ the recipient strain is crystal-negative (Cry ), .~! acquisition of a toxin pla~mid converts it to crystal !1 production ~Cry+). This method is known as conjugative plasmid transfer and is one way o~ identifying a plasmid as . ~ :
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a toxin plasmid. It has also been used to determine the toxicity and specificity of individual toxin plasmids, by ; comparing their toxin product~s) in an isogenic background.
Transconjugants (i.e. which originally may have been - 5 isogenic strains) carrying a single toxin plasmid can be used as donors in turn, and strains already carrying one or ` more toxin plasmids can be used as recipients, and can . acquire additional toxin plasmids as described in Section ~ 5.0 above and in Sections 5.6, 5.7, 5.8, 5.10 and 5.11 :: 10 below).

~. 5.2. ISOLATION OF HD-l VARIANTS
;: HD-l, a BT strain o~ varie~y kurstaki (flagellar i serotype 3ab) is the BT strain most frequently used in the U.S. to control lepidopteran pests. HD-l was subjected to ~` extensive curing manipulations in an effort to improve its . specificity and activi.ty against caterpillar pests attacking cotton plants, especially the two Heliothis sps.~ H.
virescens hereinafter referred to as HV, and H. zea hereinafter referred to as HZ. A group of HD-l variants ~` altered in plasmid content, either missing one or more :~. plasmids (e.g., partially cured), or having more complex changes in their plasmid array, was generated and bioassayed . against HV and HZ.
;: 25 Loss of individual plasmids showed that HDl-l .~ (the wild~type strain) contained two toxin plasmids, 44 and :, ~. 115 Md in size. The 115-Md plasmid coded ~or two types of ; toxin protein crystal: a bipyramidal crystal, known as Pl, . containing proteins about 130,000 d in size, and a flattened cuboidal crystal, known as P2, composed o~ protein(s) 68,000 d in size. The 115-Md pla~mid contains at least two distinct Pl toxin genes, known as the 4.5 and 60~ genes.
The 44-Md toxin plasmid coded for a Pl-type protein, distinct ~rom those coded by the 115-Md plasmid, being ~`~ 35 slightly smaller in size by approximately 2000 d.
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' ` -12- . ~3~39 The gene coding ~or this slightly smaller Pl protein is known as a 5.3-type gene. Cultures of HD-l variants lacking the 44-Md toxin plasmid made bipyramidal (P1) crystals smaller than those made by HD-1 variants carrying both toxin plasmids (and therefore containing a larger number of P1 genes). On the other hand such strains made P2 crystals which were noticeably larger than those in strains carrying the smaller Pl toxin plasmid. Therefore, strains carrying only one toxin plasmid, ~he 115-~d, made .! 10 larger quantities of P2 toxin, and had greater ratios of P2 vs. Pl produced. Electrophoresis of the crystals from a large group of ~D-1 variants confirmed the microscopic observation of the increasing size of the P2.
In FIG. 1, toxin proteins from strains carrying 15 both toxin plasmids (HDl-l, -3, -5, -7, -26) or the 115~Md plasmid only (HDl-2, -11, -12, -14, -27, -30) have been electrophoresed and resolved according to size. Equal ~,, amounts of cultures, grown under identical conditions, were ~ loaded on the gel. The~strains carrying only the 115-Md ;: 20 toxin plasmid show an approximately 50% reduction in the intensity o~ the P1 band, reflecting the 105s of the Pl ~ toxin gene(s) on the 44-Md pla~mid. The P2 protein band, - however, showed a 50-100% rise in intensity, caused by the increase in yield of P2 protein in these strains.
Some of the derivatives in FIG. 1 had undergone ~ more radical alterations than plasmid curing; in HD1-15, -.~ 18, -19, -21, and -23, the 44-Md plasmid was lost, and then :~ one of the Pl toxin genes (the ~6.6~ gene) on the 115-Md plasmid was spontaneously deleted, so that these derivatives 30 have only two active toxin genes, a 4.5-type Pl gene and a ~: P2 gene. Microscopic observation, confirmed by the gel in FIG. 1, show that cells of this strain produce Pl and P2 proteins in roughly equal amounts.

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5.3. ISOLATION OF HD269-2 (EG2069~
The BT strain HD-269 was obtained from the U.S.D.A. as a mixed culture o~ two closely-related variants.
The existence of these variants was unknown at the time of -~ 5 receipt of the culture. Both of thes~ variants were isolated, characterized (as with the HD-l variants) and established as a biologically pure culture. One variant, HD269-1 (EG2068) conkained ~wo toxin plasmids, of sizes 110 Md and 69 Md. The other variant, HD269-2 (EG2069), was a partially-cured derivative of HD269-1 and lacked the 69-Md ~oxin plasmid.

5.4. ISOLATION OF HD263-4 (EG2038) The ~D-263 parental strain, HD~63-1 (EG2035), ;~: 15 contains three toxin plasmids of sizes 110 Md, 60 ~d, and 44 Md. HD263-1 was grown with shaking in Difco nutrient broth at an elevated temperature (42C) overnight, then single colonies were isolated from the overnight culture. A colony that had lost the 44-Md toxin plasmid was discovered by ~ 20 random screening of single colonies on agarose gels; to :~ detect the absence of the 44-~d plasmid, and named HD263-4.
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;~.; 5.5, ISOLATION OF HD263-4-1 (EG2094) .~ The BT strain HD1-9 (EG2009) (see TABLE IV) was .~ 25 used as a donor by growing it together with recipient strain . HD73-26 in nutrient salts broth. Nutrient salts broth +l consi~ts of 0.8% Difco nutrient broth supplemented with Ng ' .:~ (to lmM), Ca~+ (to 0.7 mM) and Mn++ (to 0.05 ~M). Plasmid ,~ transfer was carried out by inoculating spores Df donor and recipient strains into nutrient ~alts broth and allowing ; the strains to grow together ~or 31 hours at 30C, with .I gentIe shaking. Afterwards, colonies of the recipient strain were selected by using streptomycin-containing plates ~ . (HD73-26 is resistant to streptomycin) and Cry colonies :,, 35 were then identified by phase contrast microscopy. In this . ~ .

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manner, the transconjugant HD73-26-4 (EG2236) was created, which acquired the 44~ ~d transmissible Pl-toxin codi~g plasmid from HD1-g. HD73-26-4 was then u~ed as a donor by inoculating its ~pores and those o~ the recipient strain HD263-4 (EG2038~ toyether into liquid M27 broth (the recipe is given in Section 6.1) and growing them together at 30C
~or 7 hours with gentl~ shaking. The transconjugant HD263-4-1 EG2094), which had acquired the 44+ Md Pl toxin plasmid from HD73-26-4, was isolated by random screening o~
recipient-type (PlP2+) colo~ies on agarose gels.

5.6. ISOL~TION OF HD263-4-5A (EG210~) ~ he BT strain HD-122A (EG2175) was used as a donor by growing it together with recipient strain HD73-26 by inoculating spores of both strains into M27 broth (composition described in Section 6.1 below) and allowing the strains to grow together for 8 hours at 30C, with gentle shaking. Afterwards, colonies of the recipient strain were selected by using streptomycin-containing plates (HD73-26 is resistant to streptomycin~ and Cry+ colonies were then identifi~d by phase contra~t microscopy. In this manner, the transconjugant HD73-26-23 (EG2255) was created, which acquired the 46+ and 5.4 Md plasmids from HD 122A.
HD73-26-23 was then used as a donor by inoculating its spores and those of the recipient strain HD263-4 (EG2038) together into M27 broth and growing them together at 30C
for 8 hours with gentle shaking. The transconjugant ~D263-4-5A (EG2101), which had acquired the 46+ Md P1 toxin ~,, .; plasmid ~rom HD73-26-23, was isolated by random screening of ,~` 30 recipient-type (PlP2+) colonies on agarose gels.
:, 5.7. ISOhATION OF HD269-2-7 (EG2348~
~,~ The BT strain HD-122A (EG2175) was used as a donor by growing it together with recipient strain HD73-26 by inoculating spores of both strains into M27 broth and : . .
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; growing them together for 8 or more hours at 30C, with gentle shaking. Afterwards, colonies of the recipient strain were selected by using streptomycin-containing plates (HD73-26 is resistant to streptomycin) and Cry+ colonies were then identified by phase contrast microscopy. In this manner, the transconjugant HD73-26-23 (EG2255) was created, which acquired the 46+ and 5.4 Md plasmids from HD-122A.
, HD73-26-23 was then used as a donor by inoculating its spores and those of the recipient strain HD269-2 (EG2069) together into M27 broth and growing them together at 30C for 15 hours with gentle shakiny. The transconjugant HD269-~-7 (EG2348), which had acquired the 46+ ~d P1 toxin plasmid from HD73-26-23, was isolated by random screening o~ recipient-type : (PlP2+) colonies on agarose gels.

:' 5.8. ISOLATION OF HD269-2-30 (EG2371) The BT strain EG2461, isolated from grain dust, was used as a donor by growing it togather with recipient strain HD73-26 by inoculating spores of both strains into M27 broth and growing them together for 9 1/2 hours at 30C, with '~ gentle shaking. Afterwards, colonies of the recipient strain '5 were selected by using streptomycin-containing plates (HD73-26 is resistant to streptomycin) and then Cry+ colonies were identified by phase contrast microscopy. In this manner, the transconjugant HD73-26-67 (EG2299) was created, which acquired the L.D.E. and 47+ Md plasmid from EG2461. HD73-26-67 was then used as a donor by inoculating its spores and ~ those of the recipient strain HD269-2 (EG2069) together into '~ M27 broth and growing them together at 30~C for 68 hours with gentle shaking. The transconjugant HD269-2-30 ~EG2371), which had acquired the L.D~E. and 47+ Md Pl toxin plasmid ., from HD73-26-67, was isolated by random screening of `~ recipient-type (PlP2+) colonies or agarose gels.
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5.9. ISOLATION OF HD279-72 (EG2157~
The HD-279 parental ~train, HD279-1 (EG2154), ~: contains three toxin plasmids o~ sizes 110 Md, 60 Md, and 44 Md. HD279-1 was grown on Luria Agar (1% Peptone, 0.5% Yeast ;. 5 Extract, 0.5% NaCl, 1.2% ag3r) at an elevated temperature (43C) for several days, then colonies derived from single ~ cells were isolated Prom the overgrown colony. A colony ., that had lost the 60-Md toxin plasmid was discovered by . random ~creening of single colonies on agarose gels and ;~- 10 named HD279-72 (EG2157).
'"'', :, 5.10. ISOLATION OF HD269-2-8 (EG2349~
.~` The BT strain HD-232B (EG2167) was used as a donor by growing it together with recipient strain HD73-26.
: 15 spores of both strai~s were inoculated into M27 broth and grown together for 8 or more hours at 30C, with gentle ` shaking. Afterwards, colonies of the recipient strain were . selected by using streptomycin-containing plates (HD73-26 is ;1 resistant to streptomycin) and Cry+ colonies were then ;.l 20 identified by phase contrast microscopy. In this manner, the transconjugant HD73-26-25 (EG2257) wa~ created, which ' "'~! acquired the 50+, L.D.E., 9.6, 5.4, and 1.4 Md plasmids from HD-232B. HD73-26-25 was then used as a donor by inoculating its spores and those of the recipient strain HD269-2 .. 25 (EG2069~ together into M27 broth and growing them together ~ ........................................................ .
~`, at 30~C for 16 hours with gentle shaking. The transconjugant HD269-2-8 (EG2349), which had acquired the , ., +
.. ~ 50 Md Pl toxin plasmid, and also the 9.6 Md and 1.4 Md plasmids and L.D.E. from HD73-26-25, and had lost the 7.5 Md plasmid native to HD269-2, was isolated by random screening .. ~. of recipient-type (PlP2+) colonies on agarose gels.
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5.11. IS0LATION OF HDl 19-8 (EG2397) The BT strain HD-137A (EG2161) was used as a donor by yrowing it together with recipient strain ~D73-26.
Spores o~ both ~trains were inoculated into M27 broth and .-~ 5 grown together for 8 or more hours at 30C, with gentle shaking. Afterwards, colonies of the recipient strain were selected by using streptomycin~containing plates (HD73-26 is resistant to streptomycin) and Cry+ colonies were then identified by phase contrast ~icroæcopy. In this manner, . 10 the transconjugant HD73-26-34 (EG2266) was created which ac~uired the 42+ Md plasmids from HD-137A. HD73-26-34 was i then used as a donor by inoculating its spores and those of the recipient strain HDl-l9 (EG2019) together into M27 broth ,~
and growing them together at 30C for 7 hours with gentle :, 15 shaking. The transconjugant HD1-19-8 (EG2397), which had ;~ acquired the 42+ Md Pl toxin plasmid from HD73-26-34, was : isolated by random screening of recipient-type (PlP2+) . colonies on agarose gels.
.~c .`. 20 5.12. SU~MARY OF ISOLATION AND CONSTRUCTION
~; OF NOVEL ~T STRAINS
: ., The origins and plasmid contents of several ri strains related to this invention, including partially-cured and transconjugant derivatives of HD-263 and HD-269, are .~,:`` 25 described in TABLE I, some of which strains are deposited at ., the NRRL.

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~" HD73-1 (~G2180): Prototype strain, var. kurstaki, from France.
. Plasmlds: 50, 50, 7.5, 5.4, 5.2, 4.9 Nd ~ ~.
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'~ ~oxin plasmid: 50 (P1) : HD73-26 ~EG2205): Derived from prototype sitrain HD73-1 by loss of 50 1 50, 7 . 5, 5 . d, and 5 . 2 Md pla~mids and ~ddition o~ streptomy¢in resistance.
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. I 5 Plasmidis: 4 . 9 Md Toxin plasmids: ~one (crystal negatiYe) HD263-1 (EG2035): Prototype strain, var. kurstaki, from England.
Plasimids: 130, 110, 60, 44, 43, 7.5, 5.4, 5.2, 5.0, 4.9, 1.4 Md.
~ Toxin plasmids: 110 ~Pl, P2), 60 (P1), 44 (P1) :., HD263-4 ~EG2038): Strain HD263-1 cured o~ the 44-Md toxin plasmid.
~' Toxin plasmids: 110 (Pl, P2), 60 (Pl) ~i 15 HD263-4-1 (EG2094): Transconjugant using HD263-4 as .. recipient that has acquired the 44-Md (P1) toxin `~ plasmid of HD-l.
;~ HD263-~-5A (EG2101): Traniconjugant using ~D263-4 as ': recipient that has ac~uired the 46 Md (P1) toxin plasmid of HD-122A.
~D269-1 (EG2068): Prototype strain from England, var.
~ kurstaki.
-1 Pla~imids: 130, 110, 69, 49, 44, 7.5, 5.4, :3~ 5.2, 5.0 and 4.9 Md ;1 Toxin pIasmids: 110 (P1, P2) 69 (P1) `l~ 25 HD269~2 (E~2069): Derived fro~ HD26~-1 by spontaneous loss o~ the 69 Md toxin plasmid.
~: HD269-2-7 (EG2348): Transconjugant using HD269-2 as :3 recipient that has acquired the 46 (Pl) ~d toxin plasmid from HD-122A.
HD269-2-30 (EG2371): Transconjugant using HD269-2 as recipient that ha~ acquired the 47 Md (Pl) toxin ~: plasmid from EG2461.
HD1-1 (EG2001): Prototype strain, var. kurstaki, from USA.
Plaismids: 130, ~15, 53, 51, 44, 29, 9.6, 5.4, 5.2, 4.9, 1.4 Md and-L.D.E.

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Toxin plasmids: 115 (Pl,P2), 44 (P1) HDl-9 (EG2009): Derived from prototype st~ain HD1-1 (USA) by loss of 130, 115, 51, 9.6, and 5.4 Md ~` plasmids and khe L.D.E.
Plasmids: 53, 44, 29, 5.2, 4.9, 1.4 Md " .
~. Toxin plasmido 44 (Pl) :.~ HD-122A (EG2175): Prototype ~train probably from England, '~ var. aizawai.
~,: Plasmids: 120, 110, 78/ 50, 46, 43, 33, 3~, 6.0 (O.C.), 8.0, 5.4, 4.7,~3.5 ~d and L,D.E.
Toxin plasmids: 110 ~Pl), 46(P1) : EG~461: Novel BT isolated from Kansas, U.S.A. grain dust ~, sample (whe~t).
::., 15 Plasmids: 120, 110, 47, 44, 34, L.D.E, S.0 (OC), 8.2, 3.0, 7.2, 7.0, and 3.5 Md Toxin plasmid: 110 (Pl), 47 (P1) HD279-72 (EG2157): Derived from HD279-1 by loss of the 60 i Md toxin plasmid.
c 20 HD73-26-25(EG2257): Transconjugant using HD~3-26 as :~; recipient that has acquired the 50 (P1), 9.6, ,~ 5~4, 1.4 Md plasmids and L.D.E. from ~D-232B
.0 (EG2167).
~, HD269-2-8 (EG2349): Tran~conjugant using HD26~ 2 (EG2069) ~ as recipient that has acquired the 50 (P1), 9.6,:~^i 25 and 1.4 plasmids, and ~.D.E. from HD73-26-25 (E~2257), and host ~he 7.5 ~d plasmid.
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~'i HD73-26 34 (EG2266): Transconjuqant using H~73-26 as .. recipient that has acquired the 42 ~d (P1) toxin.-; plasmid from HD-137A (EG2161).
HDl-19-8 (EG2397): ~ransconjugant using H~ 9 (EG2019) as . recipient which has acquired the 42 Md (Pl) " toxin plasmid from HD73-26-34 (EG2266~.
, nL. D. E . n i5 a linear DNA element, approx. 10 Md in size.
.~ ~oCn indicates plasmid DNA exists chiefly as open circlesO
`l " _ " indicates plasmid is a toxin plasmid ~ 35 .~

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The plasmid arrays of the novel BT strains deposited at the NRRL and those of the main precursors used : in their isolation, are shown on the gel in FIG. 2 and 4.
The pathways of construction of the novel BT strains of this invention as described in Sections 5.3-5.11 are summarized in FIGS.3 and 5.
. .
; 5.13. PRODUCTS ~ND FoRMnLATIoNs INCORPORATING BT STRAINS
BT may be used as a potent insecticidal compound -, 10 with activity against lepidopteran, dipteran, and -' coleopteran insect~. It is, therefore, within the scope of the invention that these BT strains be utilized as an ~: in~ecticide (the active ingredient) alone, or as part of a : mixture of BT with other microorgani~ms. The compositions of this invention containing these strains of BT are applied at an insecticidally effective amount which will vary depending on such factors as, for example, the specific insects to be controlled, the specific plant to be treated and method of applying the insecticidally active compositions. The preferred insecticide formulations are made by mixing BT, ~, alone or with another organism, with the desired carrier.
. The formulations may be administered as a dust or as a suspension in oil (vegetahle or mineral) or water, a '~ wettable powder or in any other material suitable for i~ 25 agricultural application, u~îng the appropriate carrier ~,` adjuvants. Suitable carriers can be solid or liquid and .. '. correspond to the substances ordinarily employed in formulation technology, e.g.~ natural or regenerated mineral substances, solvents, dispersants, wetting agents, . 30 tackifiers, binders or ~ertilizers.
~:.; The compositions of t~e invention containing BT
~,. are applied to the appropriate insect habitat at an ~ insecticidally effective amount which, as not~d abo~e, will r~ vary depending on such factors as, for example, the specific ~; 3 5 . ~ .. . ~
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insects to be controlled, the specific plant to be treated and the method of applying the insecticidally active compositions.
~arget crops (i.e~, potential habitats ~or Lepidoptera, Diptera, and Coleoptera) protected by the present invention comprise, but are not limited to, the following species of plants: cereals ~such as wheat, barley,-corn, rye, oats, rice, ~srghum, and related crops), beets, ~ cotton, leguminous plants, oil plants (such as poppy,' 10 olives, andi sunflowers) cucu~ber plants, fiber plants, ;~ citrus fruit, vegetables (such as le~tuce), deciduous trees : and conifers.
Generally stated, the preferred compositions usually contain 0.1 to 99%, preferably O.l to 95%, of the . 15 insecticidal microorganism Bacillus thuringiensis, or:i combination thereof, with other active ingredients, 1 to j` 99.9% of a solid or liquid adjuvant, and o to 25%, 's,'~,i preferably 0.1 ~o 20%, of a surfactant.
~`. The formulations containing a solid or liguid ,,~
adjuvant, are prepared in known manner, e.g., by ;i - homogeneously mixing and/or grinding the active ingredients . with extenders, e.g., solvents, solid carriers, and in some :~ cases surface active compounds (surfactants).
Suitable liquid carriers are vegetable oils, such ~i 25 as coconut oil or soybean oil, mineral oils or water. The .`,' solid carriers used, e.g., for dusts and dispersible ~ powders, are normally natural mineral ~ibers such as .~, calcite, talcum, kaolin, or attapulgite. In order to i improve the physical properties it is also possible to add `~ 30 highly dispersed silicic acid or highly dispersed`absorptive ~`~. carriers of porous typesl for example pumice, broken brick, seplolite or b8ntonite. Suitable nonsorbent carriers are s' materials such as silicate or sand. In addition, a great i .. ~

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:-`: number of pregranulated materials or inorganic or organic mixtures can be used, e.g., especially dolomite or pulveriz2d plant residues.
Depending on the nature of the ~ctive ingredients : S to be formulated, suitable surface-active ~ompounds are non-ionic, ca~ionic and/or anionic surfactants having good emulsifying, dispersing and wetting properties. The term ~surfactant~ will also be understood as comprising mixtures or surfactants.
Suitable anionic urfactants can be both water-./ soluble soaps and water-soluble synthetic surface active compounds.
~ Suitable soaps are the alkali metal salts, .:~ alkaline earth metal salts or unsubstituted ammonium salts 15 of higher ~atty acid~ (ClO-C20), e.g. the sodium or potassium salts of oleic or stearic acid, or natural fatty ., acid mixtures which can be obtained, e.g., from coconut oil :, or tallow oil. Further stable surfactants are also the ::l fatty acid methylaurin salts a~ well as modified and ~! 20 unmodified phospholipids.
:i More frequently, however, so-called synthetic ~- surfactants are of use, especially fatty sulfonates, fatty ',J ' sulfates, sulfonated benzimidazole derivatives or ~: alkylarylsulfonates.
-...... 25 The fatty sulfonates or sulfates are usually in the forms of alkali metal salts, alkaline earth metal salts :, .
~: or unsubstituted ammonium salts and generally contain a C6-. '2 C22 alkyl, e.g., the sodium or calcium salt of ~1 dodecylsulfate, or of a mixture o~ ~atty alcohol sulfate, `` 30 obtained from fatty acids. These compounds also comprise the salts of sulfonic acid esters and sulfonic acids o~
fatty alcohol/ethylene oxide adducts. The sul~onated ~. benæimidazole derivatives preferably contain two sulfonic '''!', acid groups and one fatty acid radical containing about 8 to ;~1 35 22 carbon atoms. Examples of alkylarylsulfonates are the .,~

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sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibutylnaphthalenesulfonic acid, or of a naphthalenesulfonic acid/formaldehyde condensation product. Also suitable are corresponding phosphates, e.g., salts of the phosphoric acid ester of an adduct o~ p-nonylphenol with 4 to ~4 moles of ethylene oxide.
- Nonionic surfactants are preferably polyglycol ether derivative or aliphatic or cycloaliphatic alcohol or ; lO saturated or un~aturated fatty acids and alkylphenol~, said derivative containing 3 to 10 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenol~.
Other suitable non-ionic surfac~ants are the water soluble adducts of polyethylene oxide with alkylpropylene glycol, ethylenediaminopolypropylene glycol and alkylpolypropylene glycol contain 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethylene glycol ether groups and 10 to 1000 propylene glycol ether groups.
ii ~; Representative examples of non-ionic surfactants are nonylphenolpolyethanols, castor oil, glycol ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxynethanol. Fatty acid esters of 7 25 polyoxyethylene sorbitan, such as polyoxyethylene sorbitan trioleate, are also suitable non-ionic surfactants.
Cationic surfactants are preferably quaternary ammonium salts which contain, as substituents on the nitrogen, at least one C8-C22 alkyl radical and, as further substituents, lower unsubstituted or halogenated alkyl bensyl, or hydroxylated lower alkyl radicals. The salts are preferably in the form of halides, methyl sulfates or ethylsul~ates, e.g., stearyltrimethylammonium chloride.
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6. BIOASSAYS
Bioassays are conducted by topically applying a known amount of BT suspension con~aining a known weight of BT
powder to the surface of an artificial agar-based diet. The diet is containe~ in a plastic cup and is uniform in surface axea from cup to cup. Multiple cups are treated at each treatment dose. After the liquid carrier has ~vaporated, one newly hatched larva is placed in each cup, he cup is then capped, and th~ assay is incub~ted for 7 days at 30 d~grees (centigrade) at which time mortality is recorded. The LC50 value is determined via a computer program which converts the dose-mortality data to probits and calculates the lethal concentration at which 50% of the test population would die, The protein LC50 or PLC50 is calculated by ~ultiplying the LC50 value of the ~ample by the percent of that sample which is crystal protein as d~etermined by a chemical assay.
A stock suspension o~ the BT sample is prapared by weighing 20 - 30 mg o~ the powder into a glas~ screw cap vial and adding 20 ml of 0.005% Triton X-100, The suspansion is then sonicated for about 15 seconds.
Bioas~ays generally consist of a serie~ of 8 doses with each subsequent dosQ being 1/2 or 2/3 of the previous dose. Thirty insects ar~ u~ually tested at each dose. The stock suspension is used to inoculate tha tube containing the highes~ dose. A dilu~ion series is ~hen conducted. One-hundred micro~iters of the appropriate suspension is placed on the sur~ace of each die~ cup for that dose. The liquid is spread ~venly over the diet sur~ace and after evaporation the test insec~ i~ placed on the diet surfacQ.
The BT powder may be prepared according to the rollowing s~quential procedure.
1. C~ntri~uge rinal broth or P~llicon concentrate in 500 ml cen~rifuge bottle~ ~or 20 minutas at 7000 rpm ~in JA-lO rotor). (NB. Ad~u~t pH o~ broth to 7.0 prior to centrifuging).

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2. Remove and discard supernatant.
3. Resuspend pellet i~ minimal amount o~
daionized water. Stir on magnetic stirrer for 10 minutes until homogeneous slurry is obtained.
~. Add 4-5 volumes of ac2tone.
' 5. stir acetone BUspension for 30 minutes.
; 6. Centri~uge suspension for 10 minutes at 7500 ; rpm (JA-10 rotor3.

7. Discard supernatant and resuspend pellet in :' 10 approx. 100 ml acetone.
~ 8. Stir to resuspend pellet (approx. 10 minutes :, or until an even slurry is obtained)~
, 9. Filter slurry through Whatman #l filter paper~
-;
10. Repeat steps 7-9.
~; 15 11. Transfer ~inal powder to aluminum weigh boat ~ and allow to dry overnight.
.~ 12. Weigh final powder for yield and transfer to ~ 60 ml polypropylene bottle for storage at 4C.
,.;,, l~ 20 6.1. BIOASSAY OF HD-l VARIANTS
.~ The HD-l variants were grown for bioassay as .~., ;s, follows: spores were inoculated into 5 mls of M27 broth in a i~ 50 ml sterile flask. M27 broth is composed of 33 mM each of HP04= and H2PO4- anions 9 98 mM K+; 0.17% peptone, 0.1% beef -i~ 25 extract; 150 mM NaCl; 5.5 mM glucose; 330 uM Mg , 230 uM
~` Ca++, and 17 uN Mn~ (added as the chloride salts). The ;~' cultures were incubated at 30C with shaking for 3 days, at which time sporulation znd crystal for~ation were complete.
Five ul of ste.rile 1-octanol were added as an anti-~oaming agent and the cultures were vortexed to generate a ~ homogeneous suspension, trans~erred to sterile plastic tubes, ;~) sealed, and stoxed at 5~C.

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Bioassay of thes~ liquid cultures on larvae of three species of lepidopterans revealed that different HD 1 variants had signi~icantly different toxicities, as shown in TABLE IIo ~
, TABLE II
- TOXICITIES OF HD-l DERIVATIVES AGAINST DIFFEREN~
SPECIES OF LEPIDOPTER~NS
Toxicity (PLC50) a~ainst ,` _ HDl-2 10778 165 HDl-12 104117 157 ,; 15 .~ H~ = ~. zea; TN = T. ni; SE = S. exigua; NA = not available) ~, (PLC50 is in ng o~ preparation/600 mm of diet surface.) ., , _ l 20 :.' HD-l derivatives such as HDl-12 were used as recipients in conjugative matings, during which the recipient .i strain cells would acquire new toxin plasmids from thc donor :', strain. In this way, transconjugants of HDl 12 were obtained :, 25 that contained both the 115-~d native toxin plasmid of HDl-12, and one or more toxin plasmids originating in other strains of BT. It was hoped that æome of these transconjugant strains, harboring novel combina~ions of toxin plasmids, would make toxin crystals of improved toxicity '~ 30 relative to those o~ HD1 1 (the original, parental strain), measured on a protein basis as le~hal concentration per ~`~ nanoqram (ng) o~ toxin protein. This in ~act, turned out to :~ be the case as shown in TABLE III.
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27- ~328239 `, .

. _ TABLE III
VARIATIONS IN TOXICITY OF HDl-12 TRANSCONJUGANTS IN
WHICH THE 44-MD TOXIN PL~SMID IiS R~PLAC D BY A TOXIN
, PLRSMID FROM ~NOTHER ~T STRAIN

: Strain ~V _HZ
HD1~1 (wild type) 20 140 HDl-l2-s 6 142 .~ HDl 12-11 25109 ' 10 HDl-12-12 23 91 :i HDl-12-13 14 90 .~l HDl-12-14 7 49 HDl-12-15 17 85 HDl-12-17 21113 HDl-12~18 23161 HDl-12-19 10135 ~; 15 HD1-12-20 9204 (PLC50 is in ng of preparation/600 mm2 of diet surface) . . . ~. .
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Table III lists the pLC50 (concentration of toxin 20 protein killing 50% of the test insects) on HV and ~Z of 12 transconjugants of HDl-12, HD1-12-9 through ~Dl-12-20, each :' of which carries a different new toxin plasmid (~rom 12 ,i different donor strains). Against HV, the toxicities rang~
. from a little worse than HD1-1 (HDl-12-11) to over three `1 25 times as toxic as HDl-l (HDl-12-9). A~ainst HZ, these transconjugants ranged from not as toxic as HD1~1 (HDl-12-20) `` to over twice as toxic (H~1-12-14). There are two impor~ant :~ conclusions that may be drawn from these data~ A BT strain '' can be improved (or made worse) against a given insect by ,:, 30 plasmid curing and/or plasmid acquisition. In addition, a ;~, certain degree of targeting is possible; of the 12 ~' transconjugants present~d in TA3LE III, ~Dl-12~9 is highly :,i.
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toxic against EV but no better than HD1-1 against HZ.
Conversely, some of the transconjugants (~uch as HD1-12-15) were better than HDl-l against HZ, but no better than HD1-1 against HV.
Results comparable to those obtained with HDl-1, its variants and transconjugants, have also been obtained with 8T strain HD269, that originally contained two or more toxin plasmids. Some of these results are presented in TABLE
IV~
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. ., ~ CHANGES IN ACTIVITY OF TOXIN PROTEIN (IN PLC50) .~- OF BT STRAIN HD-269 THROUGH PL~SMID
~ 15 CURING AND PLASMID TRANSFER __ _ ~ ~ s `, Strain Comment Insect pLC50 -I HD269-1 Wild type SE193 ~:l HD269-2 Cured o~ one toxin plasmid SE115 HD269-2 Cured of one toxin plasmid ~V 11 HD269-2-1 Ditto; acquired new tox. plas. HV 4 HD269~2 Cured of one toxin plasmid HZ 103 ~D269-2-1 Ditto; acquired new tox. plas. HZ 40 .:~
' PLC50 is in nq of preparation/600 mm2 of diet ~urface ;
$ 25 6.2. BIOASSAY OF BT STRAIN HD269-2-30 Bioassays of BT strain HD269-2-30 were carried out generally according to the procedure set forth above in 6.0 and in 6.1 utilizing two diffe~ent powder formulations. The results are set forth ~elow in Tables V and VI.
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BT strain HD269-2-30 ;, InsectP~C50 l ON 3.2 HV 2.5 HZ 13.2 .' NZ 9.9 .' SE 3709 ;~ SE 45.5 :
. TN 15.~
TN 2~.3 .
I Insects ;1 OM = Ostrinia nubilalis European cornborer :, HV = Heliothis virescens Tobacco budworm HZ = Heliothis zea Bollworm or corn earworm ~ SE = ~588~ e l9y8 Beet armyworm :-l TN = Trichoplusia ni Cabbage looper ,~ pLcso is in ng of preparation/600 mm2 of diet surface ",.,,~

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TABLE VI
; BT strain HD269-2-30 . .
InsectPLC50 ~V 4.9 ; HZ 47.1 ~, HZ 58.4 ~^ ~ S~ 62.6 SE 108.B
~'; TN 24.5 ~N 11.8 ~ i ~ HV = Heliothis virescens Tobacco budworm " HZ = Heliothis zea Bollworm or corn earworm :, SE = Spodoptera exiqua Beet armyworm `. TN = Trichoplusia ni Cabbage looper . PLC50 is in nanograms of preParation/600 mm2 of diet surface ~, ~
These results indicate tha~ BT s~rain HD269-2-30 ha varying j degree~ o~ activity a~ainst different lepidopteran insects.
.,, .~ 6.3. BIOASSAY OF BT STRAIN HD2~9-2-7 :~ii A bioassay of BT strain HD269-2-7 was carried out :ll generally according to the procedure set ~orth above in 6.0 and in 6.1 utilizing two different powder preparations. The results are set ~orth below in TABLES VII AND VIII.
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~T STRAIN HD269-2-7 Insect PLC50 LD 3.2 `,, LD 8.0 HV 2.1 ' ' HV 1. ~
HV 4.0 ~ ~Z 15~1 `.' . . HZ, 13.0 :t SE45.1 , TN13.0 Insects ~' LD = Lymantria dispar Gypsy moth HV = Heliothis virescens Tobac~o budworm H2 = Heliothis zea Bollworm or corn earworm SE = Spodoptera exiqua Beet armyworm TN = Trichoplusia ni Ca~bage looper PLC50 is in nanograms of preparation/600 mm2 O~ diet surface.

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TABLE VIII
~`. BT STRAIN HD269-2-7 '5 Insect _ PLC50 . 5 ~:~ LD 2.8 -~ HV 4.0 HV 3.3 ~V 4.3 i HZ 30.6 HZ 28.9 :. . SE 46.0 SE 66.0 SE 52.0 TN 10.5 TN 12.3 .
Ins~cts LD = Lymantria di~par Gypsy moth ~ 15 HV = Heliothis virescens Tobacco budworm :: HZ = Heliothis _ Bollworm or corn earworm SE = Spodoptera exigua Beet armyworm TN ~ Trichoplusia ni Cabbage looper PLC50 is in nanograms of preparation/600 mm2 of diet surface.

l~ These results indicate that BT strain HD269-2-7 has varying i~ degrees of activity against different lepidopteran insects.

6.4. BIOASSAY OF BT STR~IN HD269-2 ,~ 25 A bioassay of BT strain HD269-2 was carried out ;, generally according to the procedure set forth above in 6.0 ~ and in 6.1, utilizing two different powder preparations.
.~ The results are set forth below in TABLES IX and X.
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TABI E: IX

~-, Insect PLC50 !
. 7 HV 1 . 7 HZ 8.3 ,~ HZ 16.4 zl HZ 9.7 ' SE 25.7 SE 39.4 . SE 57.7 :.j ` :! Insects . ~ HV = Heliothis virescens Tobacco budworm ' HZ = lleliothis zea Bollworm or corn earworm ., SE = Spodoptera exi~ua Beet armyworm 1 PLC50 is in nanograms of preparation/600~ mm2 of diet surface.
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.~ BT STRAIN HD269-2 Insect PLC50 , HV 1.3 HZ: 9. 4 ', HZ 18.3 5E 94.0 SE 88 . 0 :.- TN 15.7 :~ ~D 15.7 Insects LD = Lymantria dispar Gypsy moth HY = Heliothis virescens Tobaccc~ budworm HZ - Heliothis zea Bollwonn or corn earworm SE - Spodoptera exigua B~et a~yworm TN = Trichoplusia nI Cabbage looper LC50 is in nanogram~ of preparation/600 m~2 of diet surface.

i, Th~se result~ indicate that B~ strain HD269-2 haR varying degrees o~ ac~ivity agains~ di~ferent lepidopterar~ insects.

6. 5. BIOASSAY O~ BT STRAINS HDl-19-8, ::-, HD279-72 AND HD269~2-8 A bioassay of the novel strains~ HDl-19-8, E~D279-72 ~nd HD269-2-9 wa~ carried out generally according to the ~' prc~cedure set forth above in 6. 0 and 6~1, and co~pared with ~. tha toxicity of str~ins HD1 S-1980 (the int~rnational .. , standard of commercial BT preparations) and DIPEL 2X, a commercially available E~T preparation (Abbott ~aboratories, ',1 ~, Chicago, IL).
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BT STRAINS HDl-19-8, HD279-72, and HD269-2-8 strain HV HZ SE TNLD
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HDl-lg-8 - 104 25 29 ~ All data from powder samples.
--, HV = Heliothis virescens HZ = Heliothis zea SE = Spodoptera exlgua TN = Trichoplusia ni LD = Lymantria dispar PLC50 is in nanoyrams of preparation/ÇOO mm2 of diet surface.
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; These results show that these novel strains have ,~, generally improved toxicity relative to the known strain and the commercial preparation.

7. DEPOSIT OF MICROORGANISMS
$ It is within the scope of this invention that both sporulating and nonsporulating ~Orm5 of the isolated strains oP BT microorganisms are encompassed herein. Exemplary of ~, the microorganisms usePul in the compositions and methods disclosed herein are the following Bacillus thurinqiensis , 1 .,,j ~, ~ :, ..~, ~ 1 i ~, :., , ! .
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strains which have been deposited with the Agricultural Research Culture Collection (NR~L), Peoria, IL and which have been assigned ths listed accession numbers:
.
B. thurinqiensis strainAccession Numbers -~ HD263-4-5A B-18206 :~ HD269-2-8 B-18346 , Aspects of the present invention are not to be limited in scope by the microorganisms deposited, since the deposited embodiments are intendad as individual illustrations. Indeed, various modifications of the invention in addition to those shown and described herein . will become apparent to those skilled in the art from the i foregoing description and accompanying drawings.
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Claims (10)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A Bacillus thuringiensis bacterium deposited with NRRL, and assigned an accession number, characterized in that the bacterium is selected from the group consisting of bacteria having the following accession numbers:

2. An insecticide composition characterized in that it comprises at least one of the bacteria of claim 1, in combination with a suitable carrier.

3. The insecticide of claim 2 characterized in that the carrier is a liquid carrier.

4. The insecticide of claim 2 characterized in that the carrier contains is a solid carrier.

5. A method for producing a transconjugant Bacillus thuringiensis strain having selective insecticidal activity characterized by:
(a) mixing and culturing a first Bacillus thuringiensis strain having a specific insecticidal activity conferred by a transferable plasmid with a gene coding for an insecticidal toxin protein in admixture with an intermediate Bacillus thuringiensis recipient strain, whereby the intermediate Bacillus thuringiensis recipient strain acquires by conjugation the plasmid conferring the specific insecticidal activity and thereby becomes a first transconjugant;
(b) isolating and identifying the first transconjugant Bacillus thuringiensis strain which has acquired the plasmid conferring the specific insecticidal activity;
(c) mixing and culturing the first transconjugant Bacillus thuringiensis strain isolated in step (b) in admixture with a second Bacillus thuringiensis strain that is a cured derivative of Bacillus thuringiensis strain HD263-1 or strain HD269-1 or strain HD-1, which strain has been cured of at least one plasmid, whereby the second Bacillus thuringiensis strain acquires by conjugation the plasmid conferring the specific insecticidal activity from the first transconjugant Bacillus thuringiensis strain; and (d) isolating and identifying the second transconjugant Bacillus thuringiensis strain with the specific insecticidal activity from the culture admixture of step (c).

6. The method of claim 5 characterized in that the first Bacillus thuringiensis strain, prior to the conjugation of step (a), has been cured of at least one plasmid.

7. The method according to claim 6 wherein the first, cured Bacillus thuringiensis strain has the identifying characteristics of a strain having accession number NRRL B-18207.

8. A method for producing a Bacillus thuringiensis strain having improved specific insecticidal activity, the activity being conferred by a specified toxin-encoding plasmid characterized by providing a first Bacillus thuringiensis strain possessing the specified toxin-encoding plasmid and at least one other toxin-encoding plasmid, and curing the first strain of at least one toxin-encoding plasmid other than the specified plasmid by heating the first strain to a temperature of about 37°C to about 45°C, whereby a second, cured Bacillus thuringiensis strain having improved insecticidal activity over the first strain is produced.

9. A method according to claim 8 wherein the second, cured Bacillus thuringiensis strain has the identifying characteristics of a strain having accession number NRRL B-18345.

10. A method for the control of lepidopteran insects which comprises applying to a host plant for such insects an insecticidally effective amount of a Bacillus thuringiensis bacterium selected from the group of cured and transconjugant bacteria having the following accession numbers: